Mechanisms underlying axon development are fundamental to the establishment of a functional neural circuitry, and impairments in axon growth and guidance contribute to many neurological disorders. Although many studies have shown that developing axons translate mRNAs, and that this can play roles in axon growth and guidance, the mechanisms underlying RNA-based regulation of axon development are not fully understood. This proposal is to investigate the roles of microRNAs (miRNAs) in axon development, using sensory neurons as a model system. MiRNAs are endogenous ~21 nt small RNAs that provide widespread regulation of gene expression, generally by silencing target mRNAs. In the nervous system, miRNAs function to regulate gene expression in a variety of processes such as neurogenesis, differentiation, proliferation, survival, and synaptic maintenance. However, little is known about the identity or function of miRNAs in development of the axon. In preliminary studies, disruption of the miRNA processing enzyme, Dicer, resulted in axon defects including reduced growth rate, loss of guidance cue responsiveness, and disorganization of the axon cytoskeleton. Additionally, loss of Dicer in sensory neurons resulted in the upregulation of putative miRNA target genes with well-established roles in axon growth and guidance. The Specific Aims of this proposal are 1) to identify miRNAs and their targets in developing axons, and 2) to evaluate the influence of select miRNA on axon growth and guidance during development. Experiments in Aim 1 will use conditional Dicer knockout mice, gene expression profiling, and computational approaches to identify miRNAs and Dicer- dependent target mRNAs relevant to axon development. Experiments in Aim 2 will focus on the functional analysis of individual miRNAs in the developing axon using in vitro and in vivo loss- and gain-of-function studies. Knockdown of select miRNA and gene expression profiling will reveal target mRNAs that are regulated downstream of miRNAs. Identifying miRNAs and examining their function will advance our understanding of RNA- based mechanisms in the axon, and should ultimately help to provide insight into mechanisms relevant to nerve regeneration and neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Recent evidence demonstrates that alterations in axon growth and guidance proteins are likely to play a major role in nerve regeneration and neurodegenerative disorders such as Amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Understanding how miRNAs influence the expression of mRNAs implicated in axon growth and guidance is expected to impact molecular therapeutic strategies for these serious disorders.